Human parainfluenza viruses (HPIV), HPIV1, HPIV2, and HPIV3 are significant causes of bronchiolitis, croup and pneumonia in infants and children. Karron et al., J. Infect. Dis. 172: 1445-50 (1995); Collins et al. “Parainfluenza Viruses”, p. 1205-1243. In B. N. Fields et al., eds., Fields Virology, 3rd ed, vol. 1. Lippincott-Raven Publ., Philadelphia (1996); Murphy et al., Virus Res. 11:1-15 (1988). Infections by these viruses result in substantial morbidity in children less than 3 years of age, and are responsible for approximately 20% of hospitalizations among young infants and children for respiratory tract infections.
Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV strain, nor for ameliorating HPIV related illnesses. To date, only two live attenuated PIV vaccine candidates have received particular attention. One of these candidates is a bovine PIV (BPIV) strain that is antigenically related to HPIV3, and which has been shown to protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in human infants and children (Karron et al., J. Inf. Dis. 171:1107-14 (1995a); Karron et al., J. Inf. Dis. 172:1445-1450, (1995b)). A second PIV3 vaccine candidate, JS cp45 is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (Karron et al., (1995b), supra; Belshe et al., J. Med. Virol. 10:235-42 (1982)). This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold-adaptation (ca), and attenuation (att) phenotypes which are stable after viral replication in vivo. The cp45 virus is protective against human PIV3 challenge in experimental animals and is attenuated, genetically stable, and immunogenic in seronegative human infants and children (Hall et al., Virus Res. 22:173-184 (1992); Karron et al., (1995b), supra.
HPIV3 is a member of the Paramyxovirus genus of the Paramyxovirus family, order Mononegavirales. Its genome is a single strand of negative-sense RNA 15462 nucleotides (nt) in length (Galinski et al., Virology 165: 499-510, (1988); Stokes et al., Virus Res. 25:91-103 (1992)) and encodes at least eight proteins (Collins et al., supra, (1996); Galinski, supra, (1991); Spriggs and Collins, J. Gen. Virol. 67: 2705-2719, (1986)). Three of these proteins are associated with the RNA genome to form the nucleocapsid; namely the nucleocapsid protein N, phosphoprotein P, and large polymerase subunit L. Three additional proteins are associated with the envelope, namely the matrix protein M, taught to mediate viral attachment and release, the hemagglutinin-neuraminidase protein HN, and the fusion protein F. Two other proteins, HN and F, represent the neutralizing and protective antigens of PIVs (Collins et al. In Fields Virology, 3rd ed., 1:1205-43 (1996)). Significant sequence divergence in these two protective antigens among different PIVs is the basis for the type specificity of protective immunity against these pathogens (id.).
Another protein of PIV, the C protein, is encoded by an overlapping open reading frame (ORF) of the P protein mRNA (Spriggs and Collins, 1986), and the D protein is generated by RNA editing of the P cistron (Galinski et al. Virology 186:543-50 (1992)). The P mRNA also contains an internal ORF which has the potential to encode a cystein-rich domain called V. The V ORF is also found in other paramyxoviruses and typically is accessed by RNA editing, but this is not the case with PIV. Presently, it is not known whether the PIV V ORF is expressed.
The viral genome of PIV also contains extragenic leader and trailer regions, possessing promoters required for viral replication and transcription. Thus, the PIV genetic map is represented as 3′ leader-N-P/C/D-M-F-HN-L-trailer. Transcription initiates at the 3′ end and proceeds by a sequential stop-start mechanism that is guided by short conserved motifs found at the gene boundaries. The upstream end of each gene contains a gene-start (GS) signal, which directs initiation of its respective mRNA. The downstream terminus of each gene contains a gene-end (GE) motif which directs polyadenylation and termination.
Identification of attenuating mutations in cp45 and other PIV3 vaccine candidates is of interest for a variety of reasons. In particular, it will be useful to understand the genetic basis of attenuation and to characterize the molecular virology and pathogenesis of attenuated HPIV3 strains to provide clinically acceptable vaccines for use against these and other paramyxoviruses, especially HPIV1 and HPIV2 which together account for an additional 7% of pediatric hospital admissions. To achieve these and related goals, a method for producing virus with a wt phenotype from cDNA is needed to determine which mutation(s) in the cp45 virus specify the ts, ca and att phenotypes and which gene(s) of the BPIV3 specify the attenuation phenotype.
The complete nucleotide sequences of the prototype PIV3 strain, and of the JS wt HPIV3 and cp45 strains have been determined (Stokes et al., supra., (1992); Stokes et al., Virus Res. 30: 43-52 (1993)). From these studies, the cp45 strain was shown to possess at least seventeen nucleotide substitutions compared to the parental JS wt HPIV3 strain, eight of which are correlated with changes to viral proteins. However, it has not been previously shown which of these identified mutations specify desired, e.g., ts, ca, and att, phenotypes. Recently, growth of cp45 at nonpermissive temperatures was reported to be complemented by coexpression of a cDNA clone of the L gene of the 47885 wt PIV3 strain (Ray et al., J. Virol. 70:580-584 (1996)), suggesting that the L gene may contain one or more mutations which contribute to the ts phenotype of cp45. However, the results of this study are complicated by the fact that the 47885 strain is not isogenic with the JS parent of cp45 (for example, the two viruses are 4% different at the nucleotide level, and the L proteins differ at 41 amino acid positions (Stokes et al., supra, (1992); published erratum appears in Virus Res. 27:96 (1993); Virus Res. 25:91-103. Also, this method of complementation does not provide a clear measurement of the relative contribution of the L gene mutation(s) to the overall ts phenotype of cp45.
Rescue and analysis of attenuating mutations in PIV3 and other RNA viruses require effective methods to manipulate cDNAs for the particular viruses of interest. Despite previous advancements identifying cDNAs for PIV, manipulation of the genomic RNA of this and other negative-sense RNA viruses has proven difficult. One major obstacle in this regard is that the naked genomic RNA of these viruses is noninfectious.
Successful methods for direct genetic manipulation of non-segmented negative strand RNA viruses have only recently begun to be developed (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89 (1996); Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)). Functional nucleocapsids have been successfully generated from the intracellular coexpression of separately transfected plasmids bearing the T7 RNA polymerase promoter and encoding either genomic or antigenomic RNA and the N, P, and L proteins. The intracellular cDNA expression is driven by T7 RNA polymerase which is produced by co-infecting with a vaccinia recombinant virus. This approach was first used to determine the transcription and replication requirements of cDNA-encoded minireplicons. Some success has been achieved in the application of these general methods to rescue infectious rabies virus, vesicular stomatitis virus (VSV), measles virus, and Sendai virus from cDNA-encoded antigenomic RNA in the presence of the nucleocapsid N, phosphoprotein P, and large polymerase subunit L (Garcin et al., EMBO J. 14:6087-6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81 (1995); Radecke et al., EMBO J. 14:5773-5784 (1995); Schnell et al., EMBO J. 13:4195-203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92 (1995)). Respiratory synctial virus (RSV) has also been recovered from cDNA encoded antigenome but this required the transfection of an additional plasmid encoding the M2 ORF 1 transcription elongation factor (Collins et al., 1995).
Rescue of infectious PIV virus and other Mononegavirales members is complicated by virtue of their non-segmented negative-strand RNA genome. The genomic ribonucleoprotein complexes (RNPs) of segmented genome viruses, such as influenza, are generally small in size and loosely structured, and can be assembled in vitro from RNA and required viral proteins. However, PIV and other Mononegavirales members feature much larger and more tightly structured RNPs, which tend to be refractory to functional association in vitro. Furthermore, the coding potential of HPIV3 P mRNA is complicated by cotranscriptional “RNA editing” (Galinski et al., Virology 186: 543-50 (1992)). The resultant shifts in reading frame can access internal ORFs which are expressed as chimeras fused to the N-terminal part of P. In addition, HPIV3 appears to differ from most other paramyxoviruses which express a chimeric V protein, as noted above. The corresponding set of proteins from HPIV3 editing has not yet been identified, and the internal V ORF of HPIV3 is separated from the editing site by numerous translational stop codons (Galinski et al. (1992, supra). Yet another complicating factor is that editing by BPIV3 and HPIV3 produces a novel chimeric protein D, in which the upstream half of P is fused to a domain encoded by a second internal ORF (Pelet et al., EMBO J. 10: 443-448 (1991); Galinski et al., supra, (1992)). The D protein does not have a counterpart in other paramyxoviruses.
In view of the foregoing, an urgent need exists in the art for tools and methods to engineer safe and effective vaccines to alleviate the serious health problems attributable to PIV, particularly illnesses among infants and children attributable to HPIV3. Quite surprisingly, the present invention satisfies these and other related needs.
Human parainfluenza virus type 3 (HPIV3) is a common cause of serious lower respiratory tract infection in infants and children less than one year of age. It is second only to respiratory syncytial virus (RSV) as a leading cause of hospitalization for viral lower respiratory tract disease in this age group (Collins et al., in B. N. Fields Virology, p. 1205-1243, 3rd ed., vol. 1., Knipe et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996; Crowe et al., Vaccine 13:415-421, 1995; Marx et al., J. Infect. Dis. 176:1423-1427, 1997, all incorporated herein by reference). Infections by this virus result in substantial morbidity in children less than 3 years of age. HPIV1 and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup) and also can cause severe pneumonia and bronchiolitis (Collins et al., 1996, supra). In a long term study over a 20 year period, HPIV1, HPIV2, and HPIV3 were identified as etiologic agents for 6.0, 3.2, and 11.5%, respectively, of hospitalizations for respiratory tract disease accounting in total for 18% of the hospitalizations, and, for this reason, there is a need for an effective vaccine (Murphy et al., Virus Res. 11:1-15, 1988). The parainfluenza viruses have also been identified in a significant proportion of cases of virally-induced middle ear effusions in children with otitis media (Heikkinen et al., N. Engl. J. Med. 340:260-264, 1999, incorporated herein by reference). Thus, there is a need to produce a vaccine against these viruses that can prevent the serious lower respiratory tract disease and the otitis media that accompanies these HPIV infections. HPIV1, HPIV2, and HPIV3 are distinct serotypes that do not elicit significant cross-protective immunity. The major protective antigens of PIVs are the hemeagglutinin (HN) and fusion (F) glycoproteins, which mediate viral attachment, penetration and release. Protection against reinfection is mediated primarily by virus-neutralizing antibodies.
Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV serotype, nor for ameliorating HPIV related illnesses. To date, only two live attenuated PIV vaccine candidates have received particular attention. One of these candidates is a bovine PIV (BPIV3) strain that is antigenically related to HPIV3 and which has been shown to protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in human infants and children (Karron et al., J. Inf. Dis. 171:1107-14 (1995a); Karron et al., J. Inf. Dis. 172:1445-1450, (1995b)). A second PIV3 vaccine candidate, JS cp45, is a cold-adapted mutant of the JS wildtype (wt) strain of HPIV3 (Karron et al., (1995b), supra; Belshe et al., J. Med. Virol. 10:235-42 (1982)). This live, attenuated, cold-passaged (cp) PIV3 vaccine candidate exhibits temperature-sensitive (ts), cold-adaptation (ca), and attenuation (att) phenotypes which are stable after viral replication in vivo. The cp45 virus is protective against human PIV3 challenge in experimental animals and is attenuated, genetically stable, and immunogenic in seronegative human infants and children (Hall et al., Virus Res. 22:173-184 (1992); Karron et al., (1995b), supra The most promising prospects to date are live attenuated vaccine viruses since these have been shown to be efficacious in non-human primates even in the presence of passively transferred antibodies, an experimental situation that simulates that present in the very young infant who possesses maternally acquired antibodies (Crowe et al., Vaccine 13:847-855, 1995; Durbin et al., J Infect Dis 179:1345-1351, 1999). Two live attenuated PIV3 vaccine candidates, a temperature-sensitive (ts) derivative of the wild type PIV3 JS strain (designated PIV3 cp45) and a bovine PIV3 (BPIV3) strain, are undergoing clinical evaluation (Karron et al., Pediatr Infect Dis J 15:650-654, 1996; Karron et al., J Infect Dis 171:1107-1114, 1995a; Karron et al., J Infect Dis 172, 1445-1450, 1995b). The live attenuated PIV3 cp45 vaccine candidate was derived from the JS strain of HPIV3 via serial passage in cell culture at low temperature and has been found to be protective against HPIV3 challenge in experimental animals and to be satisfactorily attenuated, genetically stable, and immunogenic in seronegative human infants and children (Belshe et al, J. Med. Virol. 10:235-242, 1982; Belshe et al., Infect Immun 37:160-5, 1982; Clements et al., J. Clin. Microbiol. 29:1175-82, 1991; Crookshanks et al., J. Med. Virol 13:243-9, 1984; Hall et al., Virus Res. 22:173-184, 1992; Karron et al., J. Infect. Dis. 172, 1445-1450, 1995b). Because these PIV3 candidate vaccine viruses are biologically derived, there is no proven methods for adjusting the level of attenuation should this be found necessary from ongoing clinical trials.
To facilitate development of PIV vaccine candidates, recombinant DNA technology has recently made it possible to recover infectious negative-stranded RNA viruses from cDNA (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89 (1996); Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), rabies virus (RaV), simian virus 5 (SV5), rinderpest virus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), measles virus (MeV), and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81 (1995); Radecke et al., EMBO J. 14:5773-5784 (1995); Schnell et al., EMBO J. 13:4195-203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92 (1995); Hoffman et al., J. Virol. 71:4272-4277 (1997); Kato et al., Genes to Cells 1:569-579, 1996, Roberts et al., Virology 247:1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; International Publication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995; U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding to published International Application No. WO 98/02530 and priority U.S. Provisional Application Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filed Jul. 15, 1996); U.S. patent application Ser. No. 09/291,894, filed on Apr. 13, 1999; U.S. Provisional Patent Application Ser. No. 60/129,006, filed Apr. 13, 1999; U.S. Provisional Patent Application Ser. No. 60/143,097, filed by Bucholz et al. on Jul. 9, 1999; Juhasz et al., J. Virol. 71:5814-5819, 1997; He et al. Virology 237:249-260, 1997; Peters et al. J. Virol. 73:5001-5009, 1999; Whitehead et al., Virology 247:232-239, 1998a; Whitehead et al., J. Virol. 72:4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; Bucholz et al. J. Virol. 73:251-259, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999, and Clarke et al., J. Virol. 74:4831-4838, 2000; each incorporated herein by reference in its entirety for all purposes).
In more specific regard to the instant invention, a method for producing HPIV with a wt phenotype from cDNA was recently developed for recovery of infectious, recombinant HPIV3 JS strain (see, e.g., Durbin et al., Virology 235:323-332, 1997; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998; U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), and U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997, each incorporated herein by reference). In addition, these disclosures allow for genetic manipulation of viral cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, e.g., which mutations in the HPIV3 cp45 virus specify its ts, ca and att phenotypes, and which gene(s) or genome segment(s) of BPIV3 specify its attenuation phenotype. Additionally, these and related disclosures render it feasible to construct novel PIV vaccine candidates having a wide range of different mutations and to evaluate their level of attenuation, immunogenicity and phenotypic stability (see also, U.S. Provisional Patent Application Ser. No. 60/143,134, filed by Bailly et al. on Jul. 9, 1999; and U.S. patent application Ser. No. 09/350,821, filed by Durbin et al. on Jul. 9, 1999; each incorporated herein by reference).
Thus, infectious wild type recombinant PIV3, (r)PIV3, as well as a number of ts derivatives, have now been recovered from cDNA, and reverse genetics systems have been used to generate infectious virus bearing defined attenuating mutations and to study the genetic basis of attenuation of existing vaccine viruses. For example, the three amino acid substitutions found in the L gene of cp45, singularly or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and attenuating mutations are present in other regions of the PIV3 cp45. In addition a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3 full-length cDNA that contains the three attenuating mutations in L. The recombinant chimeric virus derived from this cDNA is designated rPIV3-1.cp45L (Skiadopoulos et al., J Virol 72:1762-8, 1998; Tao et al., J Virol 72:2955-2961, 1998; Tao et al., Vaccine 17:1100-1108, 1999, incorporated herein by reference). rPIV3-1.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with PIV1. Yet another recombinant chimeric virus, designated rPIV3-1.cp45, has been produced that contains 12 of the 15 cp45 mutations, i.e., excluding the mutations that occur in HN and F. This recombinant vaccine candidate is highly attenuated in the upper and lower respiratory tract of hamsters and induces a high level of protection against HPIV1 infection (Skiadopoulos et al., Vaccine In press, 18:503-510, 1999).
A number of studies have focused on the possible use of viral vectors to express foreign antigens toward the goal of developing vaccines against a pathogen for which other vaccine alternatives are not proved successful. In this context, a number of reports suggest that foreign genes may be successfully inserted into a recombinant negative strand RNA virus genome or antigenome with varying effects (Bukreyev et al., J. Virol. 70:6634-41, 1996; Bukreyev et al., Proc. Natl. Acad. Sci. USA 96:2367-72, 1999; Finke et al. J. Virol. 71:7281-8, 1997; Hasan et al., J. Gen. Virol. 78:2813-20, 1997; He et al., Virology 237:249-60, 1997; Jin et al., Virology 251:206-14, 1998; Johnson et al., J. Virol. 71:5060-8, 1997; Kahn et al., Virology 254:81-91, 1999; Kretzschmar et al., J. Virol. 71:5982-9, 1997; Mebatsion et al., Proc. Natl. Acad. Sci. USA 93:7310-4, 1996; Moriya et al., FEBS Lett. 425:105-11, 1998; Roberts et al., J. Virol. 73:3723-32, 1999; Roberts et al., J. Virol. 72:4704-11, 1998; Roberts et al., Virology 247:1-6, 1998; Sakai et al., FEBS Letter 456:221-226, 1999; Schnell et al., Proc. Natl. Acad. Sci. USA 93:11359-65, 1996a; Schnell et al., J. Virol. 70:2318-23, 1996b; Schnell et al., Cell 90:849-57, 1997; Singh et al., J. Gen. Virol. 80:101-6, 1999; Singh et al., J. Virol. 73:4823-8, 1999; Spielhofer et al., J. Virol. 72, 2150-9, 1998; Yu et al., Genes to Cells 2:457-66 et al., 1999; U.S. Provisional Patent Application Ser. No. 60/143,425, filed on Jul. 13, 1999, each incorporated herein by reference). When inserted into the viral genome under the control of viral transcription gene-start and gene-end signals, the foreign gene may be transcribed as a separate mRNA and yield significant protein expression. Surprisingly, in some cases foreign sequence has been reported to be stable and capable of expressing functional protein during numerous passages in vitro.
However, to successfully develop vectors for vaccine use, it is insufficient to simply demonstrate a high, stable level of protein expression. For example, this has been possible since the early-to-mid 1980s with recombinant vaccinia viruses and adenoviruses, and yet these vectors have proven to be disappointments in the development of vaccines for human use. Similarly, most nonsegmented negative strand viruses which have been developed as vectors do not possess properties or immunization strategies amenable for human use. Examples in this context include vesicular stomatitis virus, an ungulate pathogen with no history of administration to humans except for a few laboratory accidents; Sendai virus, a mouse pathogen with no history of administration to humans; simian virus 5, a canine pathogen with no history of administration to humans; and an attenuated strain of measles virus which must be administered systemically and would be neutralized by measles-specific antibodies present in nearly all humans due to maternal antibodies and widespread use of a licensed vaccine. Furthermore, some of these prior vector candidates have adverse effects, such as immunosupression, which are directly inconsistent with their use as vectors. Thus, one must identify vectors whose growth characteristics, tropisms, and other biological properties make them appropriate as vectors for human use. It is further necessary to develop a viable vaccination strategy, including an immunogenic and efficacious route of administration.
Among a host of human pathogens for which a vector-based vaccine approach may be desirable is the measles virus. A live attenuated vaccine has been available for more than three decades and has been largely successful in eradicating measles disease in the United States. However, the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries, and the virus contributes to approximately one million deaths per year.
Measles virus is a member of the Morbillivirus genus of the Paramyxoviridae family (Griffin et al., In “Fields Virology”, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the most contagious infectious agents known to man and is transmitted from person to person via the respiratory route (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The measles virus has a complex pathogenesis, involving replication in both the respiratory tract and various systemic sites (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996).
Although both mucosal IgA and serum IgG measles virus-specific antibodies can participate in the control of measles virus, the absence of measles virus disease in very young infants possessing maternally-acquired measles virus-specific antibodies identifies serum antibodies as a major mediator of resistance to disease (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The two measles virus glycoproteins, the hemagglutinin (HA) and fusion (F) proteins, are the major neutralization and protective antigens (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996).
The currently available live attenuated measles vaccine is administered by a parenteral route (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Both the wild type measles virus and the vaccine virus are very readily neutralized by antibodies, and the measles virus vaccine is rendered non-infectious by even very low levels of maternally-acquired measles virus-specific neutralizing antibodies (Halsey et al., N. Engl. J. Med. 313:544-9, 1985; Osterhaus et al., Vaccine 16:1479-81, 1998). Thus, the vaccine virus is not given until the passively-acquired maternal antibodies have decreased to undetectable levels. In the United States, measles virus vaccine is not given until 12 to 15 months of age, a time when almost all children are readily infected with the measles virus vaccine. In the developing world, measles virus continues to have a high mortality rate, especially in children within the latter half of the first year of life (Gellin et al., J. Infect. Dis. 170, S3-14, 1994; Taylor et al., Am. J. Epidemiol. 127:788-94, 1988). This occurs because the measles virus, which is highly prevalent in these regions, is able to infect that subset of infants in whom maternally-acquired measles virus-specific antibody levels have decreased to a non-protective level. Therefore, there is a need for a measles virus vaccine that is able to induce a protective immune response even in the presence of measles virus neutralizing antibodies with the goal of eliminating measles virus disease occurring within the first year of life as well as that which occurs thereafter. Given this need, there have been numerous attempts to develop an immunization strategy to protect infants in the latter half of the first year of life against measles virus, but none of these strategies has been effective to date.
The first strategy for developing an early measles vaccine involved administration of the licensed live attenuated measles virus vaccine to infants about six months of age by one of the following two methods (Cutts et al., Biologicals 25, 323-38, 1997). In one general protocol, the live attenuated measles virus was administered intranasally by drops (Black et al., New Eng. J. Med. 263, 165-169; 1960; Kok et al., Trans. R. Soc. Trop. Med. Hyg. 77:171-6, 1983; Simasathien et al., Vaccine 15:329-34, 1997) or into the lower respiratory tract by aerosol (Sabin et al., J. Infect. Dis. 152:1231-7, 1985), to initiate an infection of the respiratory tract. In a second protocol, the measles virus was given parenterally but at a higher dose than that employed for the current vaccine. The administration of vaccines that can replicate on mucosal surfaces has been successfully achieved in early infancy for both live attenuated poliovirus and rotavirus vaccines (Melnick et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 655-712. 2 vols. Lippencott-Raven Publishers, Philadelphia, 1996; Perez-Schael et al., N. Engl. J. Med. 337, 1181-7, 1997), presumably because passively-acquired IgG antibodies have less access to mucosal surfaces than they do to systemic sites of viral replication. In this situation, the live attenuated poliovirus vaccine viruses are able to infect the mucosal surface of the gastrointestinal tract or the respiratory tract of young infants, including those with maternal antibodies, resulting in the induction of a protective immune response.
Therefore, a plausible method is to immunize via the respiratory tract of the young infant with the live attenuated measles virus vaccine, since this is the natural route of infection with the measles virus. However, the live attenuated measles virus that is infectious by the parenteral route was inconsistently infectious by the intranasal route (Black et al., New Eng. J. Med. 263:165-169, 1960; Cutts et al., Biologicals 25, 323-38, 1997; Kok et al., Trans. R. Soc. Trop. Med. Hyg. 77:171-6, 1983; Simasathien et al., Vaccine 15:329-34, 1997), and this decreased infectivity was especially apparent for the Schwartz stain of measles virus vaccine which is the current vaccine strain. Presumably, during the attenuation of this virus by passage in tissue culture cells of avian origin, the virus lost a significant amount of infectivity for the upper respiratory tract of humans. Indeed, a hallmark of measles virus biology is that the virus undergoes rapid changes in biological properties when grown in vitro. Since this relatively simple route of immunization was not successful, a second approach was tried involving administration of the live virus vaccine by aerosol into the lower respiratory tract (Cutts et al., Biologicals 25, 323-38, 1997; Sabin et al., J. Infect. Dis. 152:1231-7, 1985).
Infection of young infants by aerosol administration of measles virus vaccine was accomplished in highly controlled experimental studies, but it has not been possible to reproducibly deliver a live attenuated measles virus vaccine in field settings by aerosol to the young uncooperative infant (Cutts et al., Biologicals 25, 323-38, 1997). In another attempt to immunize six-month old infants, the measles vaccine virus was administered parenterally at a 10- to 100-fold increased dose (Markowitz et al., N. Engl. J. Med. 322:580-7, 1990). Although high-titer live measles vaccination improved seroconversion in infants 4-6 months of age, there was an associated increase in mortality in the high-titer vaccine recipients later in infancy (Gellin et al., J. Infect. Dis. 170:S3-14, 1994; Holt et al., J. Infect. Dis. 168:1087-96, 1993; Markowitz et al., N. Engl. J. Med. 322:580-7, 1990) and this approach to immunization has been abandoned.
A second strategy previously explored for a measles virus vaccine was the use of an inactivated measles virus vaccine, specifically, a formalin inactivated whole measles virus or a subunit virus vaccine prepared from measles virus (Griffin et al., In “Fields Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds.), Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). However, the clinical use of the vaccines in the 1960's revealed a very serious complication, namely, that the inactivated virus vaccines potentiated disease rather than prevented it (Fulginiti et al., JAMA 202:1075-80, 1967). This was first observed with formalin-inactivated measles virus vaccine (Fulginiti et al., JAMA 202:1075-80, 1967). Initially, this vaccine prevented measles, but after several years vaccinees lost their resistance to infection. When subsequently infected with naturally circulating measles virus, the vaccinees developed an atypical illness with accentuated systemic symptoms and pneumonia (Fulginiti et al., JAMA 202:1075-80, 1967; Nader et al., J. Pediatr. 72:22-8, 1968; Rauh et al., Am. J. Dis. Child 109:232-7, 1965). Retrospective analysis showed that formalin inactivation destroyed the ability of the measles fusion (F) protein to induce hemolysis-inhibiting antibodies, but it did not destroy the ability of the HA (hemagglutinin or attachment) protein to induce neutralizing antibodies (Norrby et al., J. Infect. Dis. 132:262-9, 1975; Norrby et al., Infect. Immun. 11:231-9, 1975). When the immunity induced by the HA protein had waned sufficiently to permit extensive infection with wild type measles virus, an altered and sometimes more severe disease was seen at the sites of measles virus replication (Bellanti, Pediatrics 48:715-29, 1971; Buser, N. Engl. J. Med. 277:250-1, 1967). This atypical disease is believed to be mediated in part by an altered cell-mediated immune response in which Th-2 cells were preferentially induced leading to heightened disease manifestations at the sites of viral replication (Polack et al., Nat. Med. 5:629-34, 1999). Because of this experience with nonliving measles virus vaccines and also because the immunogenicity of such parenterally-administered vaccines can be decreased by passively-transferred antibodies, there has been considerable reluctance to evaluate such vaccines in human infants. It should be noted that disease potentiation appears to be associated only with killed vaccines.
Yet another strategy that has been explored for developing a vaccine against measles for use in young infants has been the use of viral vectors to express a protective antigen of the measles virus (Drillien et al., Proc. Natl. Acad. Sci. USA 85:1252-6, 1988; Fooks et al., J. Gen. Virol. 79:1027-31, 1998; Schnell et al., Proc. Natl. Acad. Sci. USA 93:11359-65, 1996a; Taylor et al., Virology 187:321-8, 1992; Wild et al., Vaccine 8:441-2, 1990; Wild et al., J. Gen. Virol. 73:359-67, 1992). A variety of vectors have been explored including poxviruses such as the replication-competent vaccinia virus or the replication-defective modified vaccinia virus Ankara (MVA) stain. Replication-competent vaccinia recombinants expressing the F or HA glycoprotein of measles virus were efficacious in immunologically naive vaccinees. However, when they were administered parenterally in the presence of passive antibody against measles virus, their immunogenicity and protective efficacy was largely abrogated (Galletti et al., Vaccine 13, 197-201, 1995; Osterhaus et al., Vaccine 16:1479-81, 1998; Siegrist et al., Vaccine 16:1409-14, 1998; Siegrist et al., Dev. Biol. Stand. 95:133-9, 1998).
Replication-competent vaccinia recombinants expressing the protective antigens of RSV have also been shown to be ineffective in inducing a protective immune response when they are administered parenterally in the presence of passive antibody (Murphy et al., J. Virol. 62:3907-10, 1988a), but they readily protected such hosts when administered intranasally. Unfortunately, replication -competent vaccinia virus recombinants are not sufficiently attenuated for use in immunocompromised hosts such as persons with human immunodeficiency virus (HIV) infection (Fenner et al., World Health Organization, Geneva, 1988; Redfield et al., N. Engl. J. Med. 316, 673-676, 1987), and their administration by the intranasal route even to immunocompetent individuals would be problematic. Therefore they are not being pursued as vectors for use in human infants, some of whom could be infected with HIV.
The MVA vector, which was derived by more than 500 passages in chick embryo cells (Mayr et al., Infection 3:6-14, 1975; Meyer et al., J. Gen. Virol. 72:1031-1038, 1991), has also been evaluated as a potential vaccine vector for the protective antigens of several paramyxoviruses (Durbin et al., J. Infect. Dis. 179:1345-51, 1999a; Wyatt et al., Vaccine 14, 1451-1458, 1996). MVA is a highly attenuated host range mutant that replicates well in avian cells but not in most mammalian cells, including those obtained from monkeys and humans (Blanchard et al., J. Gen. Virol. 79:1159-1167, 1998; Carroll et al., Virology 238:198-211, 1997; Drexler et al., J. Gen. Virol. 79, 347-352, 1998; Sutter et al., Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851, 1992). Avipox vaccine vectors, which have a host range restriction similar to that of MVA, also have been constructed that express measles virus protective antigens (Taylor et al., Virology 187, 321-8, 1992). MVA is non-pathogenic in immunocompromised hosts and has been administered to large numbers of humans without incident (Mayr et al., Zentralbl Bakteriol [B] 167, 375-90, 1978; Stickl et al., Dtsch. Med. Wochenschr. 99:2386-92, 1974; Werner et al., Archives of Virology 64, 247-256, 1980). Unfortunately, both the immunogenicity and efficacy of MVA expressing a paramyxovirus protective antigen were abrogated in passively-immunized rhesus monkeys whether delivered by a parenteral or a topical route (Durbin et al., Virology 235:323-332, 1999). The immunogenicity of DNA vaccines expressing measles virus protective antigens delivered parenterally was also decreased in passively-immunized hosts (Siegrist et al., Dev. Biol. Stand. 95:133-9, 1998). Replication-defective vectors expressing measles virus protective antigens are presently being evaluated, including adenovirus-measles virus HA recombinants (Fooks et al., J. Gen. Virol. 79:1027-31, 1998). In this context, MVA recombinants expressing parainfluenza virus antigens, unlike replication-competent vaccinia virus recombinants, lacked protective efficacy when given by a mucosal route to animals with passively-acquired antibodies, and it is unlikely that they, or the similar avipox vectors, can be used in infants with maternally-acquired measles virus antibodies.
Based on the reports summarized above, it appears unlikely that a replication-competent or replication-defective poxvirus vector, or a DNA vaccine, expressing a measles virus protective antigen will be satisfactorily immunogenic or efficacious in infants possessing passively-acquired maternal measles virus-specific antibodies.
A recently developed replication-competent virus vector expressing measles virus HA that replicates in the respiratory tract of animal hosts has been developed, namely, vesicular stomatitis virus (VSV), a rhabdovirus which naturally infects cattle but not humans (Roberts et al., J. Virol. 73:3723-32, 1999; Schnell et al., Proc. Natl. Acad. Sci. USA 93:11359-65. 1996a). Since VSV is an animal virus that can cause disease in humans, development of this recombinant for use in humans will require that a VSV backbone that is satisfactorily attenuated in human infants be first identified (Roberts et al., J. Virol. 73:3723-32, 1999), but such clinical studies have not been initiated.
Although there have been numerous advances toward development of effective vaccine agents against PIV and other pathogens, including measles, there remains a clear need in the art for additional tools and methods to engineer safe and effective vaccines to alleviate the serious health problems attributable to these pathogens, particularly among young infants. Among the remaining challenges in this context is the need for additional tools to generate suitably attenuated, immunogenic and genetically stable vaccine candidates for use in diverse clinical settings against one or more pathogens. To facilitate these goals, existing methods for identifying and incorporating attenuating mutations into recombinant vaccine strains and for developing vector-based vaccines and immunization methods must be expanded. Surprisingly, the present invention fulfills these needs and provides additional advantages as described herein below.